Approximately one-third to one-half of all human pharmaceutical products currently under development and in clinical trials are derived from humanised monoclonal antibodies (mAbs). Monoclonal antibodies are ideal therapeutic candidates as they mimic natural processes of the body and are, in principle, devoid of the intrinsic toxicity often present with the use of small-molecule drugs. The global shortfall of available biomanufacturing capacity is becoming a critical limitation in mAb commercialisation. Improvements are required in all areas of the pharmaceutical supply chain, particularly downstream processing, to manage these manufacturing challenges.
Antibodies are glycoprotein molecules and belong to a class of biomolecules known as the immunoglobulins, of which there are five major classes, IgG, IgA, IgM, IgD and IgE. They are separated into these classes according to their heavy chain components and each have characteristic biological and structural properties. Antibodies are a significant part of the immune system and are produced by B lymphocytes in response to a foreign molecule (antigen). Each B lymphocyte cell will only produce a single antibody molecule specific for one binding site (epitope) on the antigen. Polyclonal antibodies are produced from more than one B lymphocyte cell clone and collectively bind to several sites on a single antigen. Each monoclonal antibody is produced by a single clone of plasma cell and are specific for a single site on the antigen.
The G class of immunoglobulins (IgG) are the most common in serum and have a molecular mass of ca. 150 kDa. Furthermore, they can be divided into four subclasses (G1-4) in humans. IgG is composed of four polypeptide chains (2 identical heavy chains and 2 identical light chains) joined by disulfide bridges to form a Y shape structure. Immunoglobulin G can be divided into two regions. The ‘tail’ of the antibody is referred to as Fc (fragment crystallisable) while the other region contains two identical fragments termed Fab (fragment antigen binding). Furthermore, each of the 4 polypeptide chains contains a constant region (CH or CL) and variable region (VH or VL). Formation of two identical antigen binding sites occurs when the variable regions of the heavy and light chains combine.
Monoclonal antibodies are currently being utilised and trialed as therapeutic and diagnostic agents. Diagnostically, they can be tagged with fluorescent or radioactive labels to test for aberrant biological phenomena associated, for example, with pregnancy, blood clots, cancers, heart disease, and viruses (e.g. AIDS). Therapeutically, they can be used for treatment in transplant rejection, various forms of cancer, auto-immune diseases (e.g. multiple sclerosis, rheumatoid arthritis) and infectious diseases (respiratory syncytial virus, cytomegalovirus, septicaemia). Furthermore, they can also be used in protein structure analysis, affinity purification of biomolecules and drugs.
The sources from which antibodies can be isolated include natural sources (body fluids of immunised animals or humans) and recombinant sources (supernatents of lysates from engineered cells derived from hybridoma or bacterial cells). The number of expression systems for antibody production has recently expanded and now includes mammalian cells, insect cells, yeasts, bacteria, transgenic animals and transgenic plants, all of which have their advantages and disadvantages. The therapeutic monoclonal antibodies available on the market today are derived from animal cell culture.
Antibodies need to be extracted and purified whether they are derived from natural sources or recombinant cells. Although generic purification schemes have been suggested, they only serve as a guide with each purification method examined on a case-by-case basis, where it may be necessary to optimise certain steps. Differences in amino acid composition and sequence of the variable domains of monoclonal antibodies, the level and type of glycosylation, and the nature of any other post-translational or chemical modification (e.g. pegylation) have a profound effect on the chemical, physical and biological properties of the antibody. Antibodies vary in their isoelectric point, solubility and resistance to extremes of pH which complicates the use of a generic purification system.
Since polyclonal antibodies have heterogeneous specificity due to diversity in their antigen binding site, a method is required that facilitates purification of the total antibody population from a mixture. Although homogeneous in terms of specificity, monoclonal antibodies similarly require robust separation methods for their purification from feedstock mixtures. In both cases, the highly conserved regions (Fc) of antibodies can be exploited for their capture and subsequent purification from complex feedstocks. As well as the host cells and cell debris, contaminants may include host cell proteins, viruses and bacterial pathogens or their breakdown products (e.g. endotoxins) and media additives (protein growth promoters and stabilizers or serum supplements). Furthermore, impurities may also consist of the expressed antibody which is miss-folded or proteolytically degraded, as well as aggregates of the desired antibody.
Because of the current dosage regimes (i.e. several hundred milligrams to a gram per dose), a large production capacity is required for the manufacture of therapeutic mAbs with hundreds of kilograms to more than one metric ton of product needed per year. The conditions and diseases to be treated often require repeat doses on a gram scale unlike small-molecule pharmaceuticals where dosing is often only in the milligram range. This is due to the large size of the mAb molecules, their mode of action and the nature of the therapies used.
The high doses of therapeutic antibodies required, has two implications:                i) they must meet stringent requirements such as proof of identity, purity, stability, potency and safety; and        ii) production cost per dose must be kept to a minimum.        
To achieve these goals, effective purification strategies and processes need to be developed and employed. A general purification scheme for mAb production can be divided into three parts: the capture step, the intermediate fractionation step and the final purification and final polishing steps. No single generic downstream process is currently available that attains all of these features. With new production methods under accelerated development, cell culture and fermentation capabilities are being increased significantly with respect to expression levels. However, the growing demand for mAbs over the coming years is expected to exceed the current worldwide production capacity. The aim of downstream processing (extraction, separation and purification) is to implement the most direct route from starting material to the desired final product. Due to the rate limiting upstream production requirements, downstream processing must be made as fast and efficient as possible. Yields need to be maximized while retaining the original biological activity of the antibody. Furthermore, when developing monoclonal antibody therapeutics, it is vital to consider the downstream processing costs as they can account for up to 80% of the overall cost of production.
Downstream processing commonly involves a range of membrane filtration steps and chromatographic unit operations. The general guidelines for these operations ensure that:                the most abundant impurities are removed early in the process;        the easiest separations are run early in the process;        the difficult and/or expensive separations are performed towards the end of the process;        separations are chosen to take advantages of differences in the properties of the antibody and contaminating impurities;        the operations are structured in an orthogonal manner to exploit different separation mechanisms.        
After cell culture or fermentation, an initial clarification step, which is required to remove whole cells and large cell debris particles, can be achieved by centrifugation or microfiltration. A second clarification step helps to clear colloidal particulate material destructive towards finer filters downstream. Bacteria and other bioburden are then eliminated by using sterilizing filters before the first chromatographic unit operation.
Chromatographic techniques for the purification of monoclonal antibodies, include hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEC), hydroxyapatite chromatography (HAC), immobilised metal affinity chromatography (IMAC) or even size exclusion chromatography (SEC) and affinity chromatographic procedures.
Affinity chromatography involves the separation of the target protein from a complex mixture based on a highly specific, reversible adsorption of the desired protein onto a chromatographic matrix. The interaction occurs between certain proteins in the mixture and a bio-specific ligand that is immobilised on the solid phase, most often a polymeric solid support material such as a polymeric gel in the form of a bead. Affinity chromatography is best suited for a capture or intermediate purification step within a purification process as it possesses high selectivity and high capacity, which enables high recovery of concentrated sample with up to several thousand fold increase in purity.
Affinity adsorbents need to be reusable and the stability of the ligand and matrix with respect to the cleaning conditions used will determine the number of cycles possible. For sanitisation, a treatment with 1M sodium hydroxide is often employed to remove impurities that remain bound after the elution step and provide a column ready for further purification cycles. Alternative regeneration methods will need to be determined and optimised if the ligand or adsorbent is not resistant to caustic treatment since this will have a material bearing on critical infrastructure protection requirements.
Affinity chromatography using immobilised Protein A is currently a commonly used method for purification of antibodies. The production and isolation of Protein A involves a complex and labour intensive procedure, making the final product very expensive. Particular care is therefore needed to preserve the column for multiple uses. Protein A is limited in its selectivity towards different classes of immunoglobulins. It will mainly bind IgG, although not all subtypes, and has no affinity towards IgE and IgY. Its binding is variable towards IgA and IgM. Antibodies are commonly eluted from Protein A affinity columns under low pH (pH 2-3) conditions, however such conditions can alter their conformation and ultimately may cause a loss in their biological activity. The Protein A—IgG interaction, occurring at the Fc fragment, can affect the antibody's local structure to a certain extent, causing destabilisation and an altered susceptibility to proteolytic attack or aggregation.
Two of the most serious issues that limit the use of Protein A are its cost and leakage into the purified antibody preparations. This can be due to its degradation by proteases in the feedstock itself, the immobilisation method used or the stability of the backbone chromatographic support material. Time consuming analytical methods are therefore needed to detect contaminants in the purified product before it can be approved for therapeutic use. Furthermore, a reduction in binding capacity of the column occurs as a result of Protein A leakage. Because the immobilised Protein A also has a low resistance when cleaning the column with caustic solutions (unlike chemical ligands), this makes its use less practical in terms of sanitisation and longevity, although Protein A structural variants which have been genetically engineered for greater pH stability are now available.
Another bacterial Fc receptor, Protein G, has specificity for different antibody classes, subclasses and species. However, the cost of this reagent is much greater than Protein A itself and this constraint has limited its commercial use, even though purification conditions are less harsh compared to those for Protein A.
Bio-specific affinity chromatography involves separation based on antibody-antibody affinity recognition, antigen-antibody affinity interaction, or bacterial Fc receptor-antibody adsorption. Monoclonal and polyclonal antibodies can be selectively purified by immobilising specific antibodies (anti-immunoglobulins) as affinity ligands on a chromatographic matrix. Due to the extreme specificity of the antibody ligand, a new adsorbent must be designed every time a new antibody requires separation. This approach involves production of the antibody's ligand, and its purification and immobilisation. All of these processes increase costs, hence this approach is currently restricted to laboratory scale applications. Because antibodies possess recognition sites for specific antigens, immobilisation of these antigens as affinity ligands is commonly used. The restrictions of this method are the availability of the antigen and difficulties to achieve elution of the antibody in high yield due to the high affinity.
In an attempt to overcome some of the drawbacks of Protein A as mentioned previously, a range of triazine-dye related mimetic compounds have been generated via combinatorial library screening or computer modelling. Lowe and co-workers synthesised Protein A mimetic ligands based around phenylalanine, tyrosine and isoleucine residues attached to a trichlorotriazine ring. The resulting ligands had millimolar affinities (KA's) for IgG, 102-104 M−1. 1 Lowe et al. also attached 4-amino-1-naphthol and 3-aminophenol to the triazine core (Ligand 22/8) and the resulting adsorbent again had suboptimal (millimolar) affinity (1.4×105 M−1) with binding capacity of 51.9 mg IgG/g moist weight gel.2,3 Other chemical or peptidic ligands have also been investigated. Thus, Fassina et al. have reported a synthetic peptide ligand, TG19318: (Arg-Thr-Tyr)-4-(Lys)-3-Gly, which is able to mimic Protein A in recognition of the Fc region of antibodies.4 Moreover, the ligand had a broader specificity than Protein A, interacting with IgG, IgA, IgM, IgE and IgY from different sources.
In 1985, Porath and co-workers described an adsorption process for the fractionation of certain proteins, i.e. immunoglobulins and α2-macroglobulins, from serum, which they termed “thiophilic adsorption”.5 This adsorption was particularly affected by the presence of high concentrations of neutral salts, using resins that were divinylsulphone (DVS)-activated and blocked with β-mercaptoethanol. A series of ligands related to β-mercaptoethanol were also screened for their protein binding ability. The general structure for a thiophilic adsorbent (T-Gel) could be represented by the structure depicted below:

Porath and Oscarsson later described a similar protein binding behaviour to that of the thiophilic adsorbent (T-Gel) when 2-mercaptopyridine was coupled to epichlorohydrin activated gels. Protein adsorption was achieved in a salt promoted manner6.

Knudsen and co-workers prepared adsorbents based on hetero-aromatic ligands immobilised on divinylsulfone-activated gel which gave them a thiophilic character. Isolation of IgG from human serum in the presence of lyotropic salts using ligands including 2-, 3-, or 4-hydroxypyridine, 2-aminopyridine, 4-aminobenzoic acid, 4-methoxyphenol and imidazole was achieved.7 Although a high binding capacity was observed, the eluted samples were still contaminated with other serum proteins.
Schwarz et al. immobilised 2-mercaptopyridine, 2-mercaptopyrimidine and mercaptothiazoline onto both epoxy-activated agarose and silica.8 The latter two ligands were chosen on the basis of their higher hydrophilicity and electron density compared to 2-mercaptopyridine. Adsorption of antibodies was achieved in the presence of sodium sulfate with dissociation constants (KD) in the mid 10−7 M range. Binding capacities were also found to be higher for the silica based adsorbents. Schwarz later prepared several adsorbents using five-membered heterocyclic rings, containing at least two heteroatoms, as ligands (see below).9 The presence of at least one double bond within the ring was required for antibody adsorption, and only a slight increase in capacity at comparable densities was achieved with two double bonds, as found in the mercaptothiazole structure.

Scholz and co-workers coupled 2-mercaptopyridine, 2-mercaptopyrimidine and 2-mercaptonicotinic acid to DVS- and epichlorohydrin-activated Sepharose.10 The adsorbents prepared by DVS-activation gave a higher recovery than the corresponding epichlorohydrin-activated adsorbents under salt-promoted conditions. The DVS-activated adsorbents immobilised with 2-mercaptopyridine and 2-mercaptonicotinic acid (see below) were additionally shown to bind immunoglobulins in a salt-independent manner and consequently desorbed with 10 mM NaOH.

Scholz et al. also immobilised MECH, 3-(2-mercaptoethyl)quinazoline-2,4(1H,3H)dione, an immunostimulatory substance described by Drössler11 to DVS-activated agarose.12 The derivatised gel was able to bind antibodies (ca. 18 mg/mL gel) from human or animal serum under low-salt conditions at pH 7.4 and elution was achieved by raising the pH with dilute alkali.

In contrast to Protein A and Protein G affinity chromatography, thiophilic chromatography has the following potential advantages:                low adsorbent cost,        broad specificity for antibodies (type and subclass) from various sources,        high binding capacity,        mild elution conditions,        greater chemical stability of the affinity ligands,        lack of ligand leakage,        ability to separate and purify (recombinant) antibody fragments lacking the Fc receptor.        
The binding of antibodies to the prior art adsorbents is, however, less specific than the binding to Protein A or G bacterial Fc receptors.
With an increasing number of therapeutic monoclonal antibodies being developed, and the lack of reliable and cost effective purification protocols currently available, there is a need for procedures that can guarantee consistency in the quality of the product. Considerable effort has thus been made towards the synthesis of low-molecular weight molecules which are able to:                i) bind antibodies with similar or enhanced affinity as found with Protein A, and/or        ii) possess improved chemical and physical properties within a process context.        
The synthetic affinity ligands used in the aforementioned thiophilic chromatographic adsorbents are also preferably endowed with characteristics that result in increased chemical and biological resistance to degradation, reduced toxicity and leakage, high antibody binding capacity and broader selectivity. Overall, improvements in these characteristics will achieve a significant reduction in antibody production costs. The present invention seeks to address one or more of these existing shortcomings.